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Home > Tumor-specific delivery of a paclitaxel-loading HSA-haemin nanoparticle for cancer treatment
Tumor-specific delivery of a paclitaxel-loading HSA-haemin nanoparticle for cancer treatment
Hea-Jong Chung, PhD, Hyeon-Jin Kim, PhD, Seong-Tshool Hong, PhD
There have been strong interests in developing nanoparticles for specific delivery of anti-cancer drugs to the targeting cancer tissues.1–3 Nanotechnology-based chemotherapy could eradicate cancers if anticancer drugs are specifically delivered to the cancers as intended. Among various materials tested as base materials for nanoparticles, polyethylene glycol (PEG) has become the most widely used base material for the formulation of nanoparticles.4–6 Doxil® (pegylated liposomal doxorubicin) and Onivyde® (pegylated liposomal irinotecan) are examples of approved nanoparticles coated with PEG.7,8 The advantage of PEG coatin\g in nanoformulation is it's resistance to phagocytosis, which causes PEG-based nanoparticles to avoid clearance by the mononuclear phagocyte system in blood after systemic administration.9–11 The resistance of PEG-based nanoparticles to phagocytosis allows them to circulate in the blood for a prolonged period, up to several hundred times that of the free drug. This prolonged circulation significantly increased accumulation of the PEG-based nanoparticles into cancers in tumor-xenografted animal models.12–14 In humans, however, there have been difficulties to reach the same level of cancer-targeting and therapeutic efficacy of PEG nanoparticles, diminishing the enthusiasm for the development of PEG-based nanoparticles. Interestingly, it has been shown that PEG actively adsorbs serum proteins in human blood to form protein coronas, which swells the PEG-based nanoparticle from a nanoscale particle to a microscale particle.15–18 It seems clear that the formation of these protein coronas in the PEG nanoparticle contributes to the loss of cancer-targeting.19,20
Despite the disappointing clinical results of a few nanoparticles, an albumin-bound form of paclitaxel, Abraxane® (Celgene), showed enhanced therapeutic efficacy than its free drug, paclitaxel.21–23 Contrast to PEG which has a chemical nature for interaction with proteins to form protein coronas on PEG-based nanoparticles, human serum albumin (HSA) does bind with any serum proteins for polymerization, eliminating the possibility of corona formation on an HSA-based nanoparticle in the blood. In fact, comparative analyses on the clinical efficacies of the nanoparticles developed to date indicated that HSA is the best base material for nanoformulation.24,25 However, the nature of HSA, such as resistance to polymerization, becomes the main problem in the fabrication of an HSA-based nanoparticle. Since HSA does not polymerize itself in aqueous solution, current paclitaxel formulation with HSA, i.e., Abraxane®, rapidly dissociate in serum without benefit of nanoformulation.24,25 Therefore, one of the current challenges in nanotechnology is to develop a nanoformulation technology allowing HSA as a base material for stable nanoparticles. In this work, we successfully developed a stable paclitaxel nanoparticle, TENPA, in which HSA is non- covalently polymerized with haemin as an adhesive agent. The structural integrity and stability of TENPA were ideal for passive targeting. Moreover, the outside components of TENPA, both HSA, and haemin, are excellent cancer-targeting moieties for active targeting. These characteristics of TENPA resulted in reduced toxicity as well as enhanced efficacy with eradication of end-stage cancer in xenografted mouse experiments.
Methods
Nanoformulation of TENPA
Eighty-eight mg of HSA (Lee Biosolutions, Maryland Heights, MO, USA) was dissolved in 10 mL of 10 mM NaCl. After adjusting the pH to 8.0, 10 mg of paclitaxel (LC Laboratories, Woburn, Massachusetts, USA) in 1 mL of ethanol was added to the HSA solution very slowly until the solution became turbid. One mg of haemin (Sigma-Aldrich, St. Louis, Missouri, USA) was added to the solution following mild heating of the final formation of TENPA. The TENPA solution was filtered through a 0.22 μm filter. The filtered TENPA was collected by centrifugation and solubilized in 1 mL of 10 mM NaCl. The detailed protocol is described in the Supplementary material.
Physical characterization of TENPA
The average diameter and size distribution of TENPA were measured by a dynamic light scattering method using Zeta Sizer (Malvern Instruments Limited, Malvern, Worcestershire, UK). The zeta potential of TENPA was determined at room temperature using a Nanotrac Wave (Microtrac, Montgomery- Ville, PA, USA). The shape and surface morphology of TENPA were investigated by transmission electron microscopy (TEM) (Hitachi, Chiyoda, Tokyo, Japan). The detailed protocol is described in the Supplementary material.
Cell culture
In this study, the human lung cancer cell lines NCI-H460 (Korea Cell Line Bank, Seoul, South Korea) and NCI-H460-luc2 (Caliper Life Sciences, Waltham, Massachusetts, USA), the human breast cancer cell lines MDA-MB-231 (Korea Cell Line Bank, Seoul, South Korea) and MDA-MB-231-luc2 (Caliper Life Sciences, Waltham, Massachusetts, USA), the PC-3 human prostate cancer cell line (Korea Cell Line Bank, Seoul, South Korea), the human SK-OV-3 ovarian cancer cell line (Korea Cell Line Bank, Seoul, South Korea), and NIH-3 T3 fibroblast cells (Korea Cell Line Bank, Seoul, South Korea) were used. NCI- H460 was used for biodistribution analysis and for the determination of LD50. For efficacy studies, NCI-H460-luc2 and MDA-MB- 231-luc2 were used. For microarray analysis, NCI-H460 MDA- MB-231, PC-3, and SK-OV-3 were used. These cell lines were maintained as monolayer cultures in RPMI-1640 (HyClone Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin) at 37 °C in a humidified incubator containing 5% CO2.
Animal studies of TENPA
Animal studies were carried out in strict accordance with the recommendations in the Guide for the Ethics Committee of Chonbuk National University Laboratory Animal Center. The protocol was approved by the Ethics Committee of the Chonbuk National University Laboratory Animal Center (Permit Number: CBU 2012–0040). All efforts were made to minimize suffering. Female nude mice with a BALB/c genetic background were purchased (Damool Sciences Co., Daejeon, South Korea) at 6 weeks of age and housed in a germ-free environment at 4 ~ 5 mice in a cage. They were maintained under a 12 h/12 h light/ dark cycle at a temperature of 22 °C and humidity of 55 ± 5%. At the start of treatment, body weights ranged from 21 to 25 g, and ages ranged from 6 to 8 weeks. The xenografted human cancer models were developed by injecting 200 μL of NCI- H460-luc2 (5 × 106 cells) or MDA-MB-231-luc2 (1 × 107
cells) into the subcutaneous region of each nude mouse. Cancer growth in each xenografted mouse was monitored daily until its volume reached approximately 50 mm3 for the early-stage cancer model or approximately 200 mm3 for the late-stage cancer model. Caliper (Mitutoyo, Takatsu-Ku, Kawasaki, Japan) measurements of the longest (L) and shortest (W) cancer diameters (mm) were used for monitoring the growth of each cancer. The formula for an ellipsoid sphere [(L × W2) / 2] was used to calculate the tumor volume. The volume was converted to tumor weight assuming unit density (i.e., 1 mm3 = 1 mg).
Biodistribution analysis of paclitaxel
The biodistribution of paclitaxel in tumors and normal organs was studied by using cancer-bearing nude (nu/nu) mice. Female BALB/c nude mice (approximately 7 weeks) were subcutaneously inoculated in the abdomen area near flank with 5 × 106 NCI-H460 lung cancer cells resuspended in 70% growth medium and 30% Matrigel. When the xenografted NCI-H460 lung cancer cells reached a size of 100 mg (15–20 days after inoculation) in nude mice, the mice (n = 3) were intravenously injected with TENPA, Abraxane or Taxol containing 20 mg/kg of paclitaxel each. After injection, each organ, cancer tissues and blood were isolated 12 h later. Each organ and cancer tissue was washed and then homogenized for 2 min in the same volume of cell lysis buffer by using a bead beater (Tokken Inc., Kashiwa, Chiba, Japan). Nine volumes of ethanol were added to the completely homogenized samples by the bead beater following centrifugation at 21,130 g for 30 min at 4 °C. Fifty milliliters of each supernatant were taken for dilution 20 times with ethanol, followed by quantitative analysis of paclitaxel using a reverse phase HPLC with Agilent 6410 Triple Quad LC/MS/MS detector (Agilent technology, Santa Clara, CA, USA).
Determination of LD50
Seven-week-old female BALB/c nude mice without cancer or with NCI-H460 lung cancer cell xenografts at a tumor size of 200 mm3 were randomly divided to each group (n = 3) and received the single indicated dose of Taxol (Bristol-Myers Squibb, New York City, New York, USA), Abraxane (Celgene, Summit, New Jersey, USA), or TENPA by intravenous injection to determine 50% mortality (LD50). The injection doses were in the ranges of 20, 40, 60, 80, 100, 120, 250, 300, 350, and 400 mg/kg, as indicated in Figure 4, B and C. The control groups received saline. The weight and physical states of all the mice were monitored for a period of 10 days. Doses that resulted in LD50 were calculated using the fitted mortality curves (GraphPad Prism, La Jolla, CA, USA).
Assessment of anti-tumor efficacy of TENPA
Seven-week-old female BALB/c nude mice were subcutaneously inoculated in the abdomen area near flank with 5 × 106 NCI-H460-luc2 lung cancer cells or 1 × 107 MDA-MB-231- luc2 breast cancer cells. After the tumor reached to the desired volume, either 50 or 200 mm3, animals were randomized into groups such that the mean tumor weights were similar between groups. Mice subsequently received I .v. injections at 3-day intervals with anticancer drugs, 10 mg/kg of paclitaxel in Taxol, Abraxane, or TENPA. PBS was used for the control group of mice. The PDT group was exposed for 30 min to LED (630 nm) immediately after TENPA injection. Cancer growth was monitored with an IVIS® imaging system (Caliper Life Sciences, Waltham, Massachusetts, USA) after injection of 100 μL of 30 mg/ml D-luciferin in PBS into each mouse. The bioluminescence intensities of each mouse were calculated from the obtained luminescence signals using the software (Living- Image® Software Perkin Elmer, Waltham, Massachusetts, USA) provided by the manufacturer. Lung cancer xenograft models were established similar to breast cancer-bearing mice, except for a lung cancer cell line, NCI-H460-luc2 was used. The nude mice with a mean tumor volume of 200 mm3 were used for the late-stage cancer model.
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